Competition between Supramolecular Interaction and Protein–Protein Interaction in Protein Crystallization: Effects of Crystallization Method and Small Molecular Bridge

We introduced a small molecular “inducing ligand” strategy to crystallize proteins via dual noncovalent interactions. Here we demonstrate that the variant protein-packing frameworks of concanavalin A (ConA) binding with ligands are controlled by different crystallization methods. Besides, the protein crystalline frameworks are also controlled by small-molecule inducing ligands <b>R</b><i><b>n</b></i><b>M</b> (<i>n</i> = 1–5), which varies in the number of ethylene oxide repeating units. To better understand the mechanism of different packing frameworks controlled by different inducing ligands, all-atom molecular dynamic (MD) simulations were performed. The MD simulation focused on the dynamic dimerization behavior of <b>ConA</b>-<b>R</b><i><b>n</b></i><b>M</b> system, which revealed clear relationships between the length of inducing ligands and ConA crystalline. In short, besides protein-packing framework control via different crystallization methods, inducing ligands <b>R</b><i><b>n</b></i><b>M</b> with suitable spacer lengths (<i>n</i> = 3, 4) also play an important role in desired and stable crystalline frameworks

Global Transcriptome Profiling of <i>Salicornia europaea</i> L. Shoots under NaCl Treatment

<div><p>Background</p><p>Soil salinity is a major abiotic stress that limits agriculture productivity worldwide. <i>Salicornia europaea</i> is well adapted to extreme saline environments with more than 1,000 mM NaCl in the soil, so it could serve as an important model species for studying halophilic mechanisms in euhalophytes. To obtain insights into the molecular basis of salt tolerance, we present here the first extensive transcriptome analysis of this species using the Illumina HiSeq™ 2000.</p><p>Principal Findings</p><p>A total of 41 and 39 million clean reads from the salt-treated (Se200S) and salt-free (SeCKS) tissues of <i>S. europaea</i> shoots were obtained, and de novo assembly produced 97,865 and 101,751 unigenes, respectively. Upon further assembly with EST data from both Se200S and SeCKS, 109,712 high-quality non-redundant unigenes were generated with a mean unigene size of 639 bp. Additionally, a total of 3,979 differentially expressed genes (DEGs) were detected between the Se200S and SeCKS libraries, with 348 unigenes solely expressed in Se200S and 460 unigenes solely expressed in SeCKS. Furthermore, we identified a large number of genes that are involved in ion homeostasis and osmotic adjustment, including cation transporters and proteins for the synthesis of low-molecular compounds. All unigenes were functionally annotated within the COG, GO and KEGG pathways, and 10 genes were validated by qRT-PCR.</p><p>Conclusion</p><p>Our data contains the extensive sequencing and gene-annotation analysis of <i>S. europaea</i>. This genetic knowledge will be very useful for future studies on the molecular adaptation to abiotic stress in euhalophytes and will facilitate the genetic manipulation of other economically important crops.</p></div

Size distribution of the contigs and unigenes generated by<b><i>de novo</i></b><b> assembly.</b>

<p>(A) Size distribution of contigs. The x-axis represents contig size, and the y-axis represents numbers of contigs of a certain length. (B) Size distribution of unigenes. The x-axis represents unigene size, and the y-axis represents the number of unigenes with a certain length. (C) Size distribution of All-Unigenes. The x-axis represents All-Unigenes size, and the y-axis represents the number of All-Unigenes with a certain length.</p

Summary of sequencing and assembly results.

<p>N50: 50% of the assembled bases were incorporated into sequence with N50 length or longer.</p><p>Mean: Mean length of assembled sequences.</p

CdpR directly binds to <i>pqsH-cdpR</i> intergenic region and regulates <i>pqsH</i> expression.

<p><b>(A)</b> CdpR bound to the <i>pqsH</i>-<i>cdpR</i> intergenic region according to the ChIP-seq analyses. <b>(B)</b> EMSA experiment showed that CdpR directly bound to the <i>pqsH</i>-<i>cdpR</i> intergenic region (<i>pqsH-p</i>) but not to that of <i>pqsR</i>. PCR products containing <i>pqsH</i> or <i>pqsR</i> promoter regions were added to the reaction mixtures at a concentration of 40 nM. The protein concentration (μM) for each sample is indicated above its lane. <b>(C)</b> CdpR bound to the motif (CTGCGCCTGGATGAT) in the <i>pqsH</i> promoter region. Electropherograms show the protection pattern of the <i>pqsH</i> promoter region after digestion with DNase I following incubation in the absence or presence of 4.0 μM CdpR. The protected region showed significantly reduced peak pattern than seen in the control. <b>(D)</b> The activity of <i>pqsH</i> was drastically activated by CdpR <i>in vivo</i>. The expression of <i>pqsH</i> was measured in the wild-type PAO1, the Δ<i>cdpR</i> mutant, and the complemented strain (Δ<i>cdpR</i>/p-<i>cdpR</i>). ***<i>p</i> < 0.001 based on Student’s <i>t</i> test. EV represents empty vector. <b>(E)</b> Western-blotting confirms that the expression of <i>pqsH</i> was drastically lower in the Δ<i>cdpR</i> strain than in the wild-type PAO1. The wild-type PAO1, the Δ<i>cdpR</i> mutant, and the Δ<i>cdpR</i> complemented strain containing the integrated single-copy plasmid CTX-<i>pqsH</i>-flag were cultured at OD₆₀₀ = 0.6 and OD₆₀₀ = 1.2. The whole-cell extracts from the designated strains were subjected to SDS/PAGE separation and subsequent immuno-blotting. EV represents empty vector. <b>(F)</b> The production of PQS was decreased in the <i>cdpR</i> mutant. Lane 1 contains 50 ng of PQS. Lanes 2–7 represent the wild-type PAO1, the Δ<i>cdpR</i> mutant, the Δ<i>cdpR</i> complemented strain, the Δ<i>pqsH</i> mutant, the Δ<i>pqsR</i> mutant, and the Δ<i>pqsA</i> mutant, respectively. EV represents empty vector.</p

Issues of the required consent to adoption

Resumé The Matter of the adoption agreement request The aim of this thesis is to offer a complex view of the matter of the adoption agreement. It may be unbelievable how significant may such an agreement be and what all is changed due to it - the position of the child being adopted, his biological parents, adoptive parents and his whole family as well. The text is systematically devided into five chapters. The introductory chapter clarifies the general aspects of the adoption process. It is devided into two parts dedicated to the clarification of the notions for example alternative family care, historical development, sources of the law dealing with adoption and social-legal child protection. The main part of the thesis is dealt with in the second chapter, it is the process of adoption agreement. It is devided into seven parts concerning the matter of adoption agreement request by adult and non-adult parents, agreement of the child being adopted and various specific cases when for example only one of the married couple or a new partner of the child's parent is adopting. The question of the so-called "straight adoption" is worth mentioning as well - litigant, nonetheless still existing practice of Fond ohrožených dětí (The Foundation for endangered children). Even the figures of custodian and guardian play..

Validation of Transcriptome Profiling expression with QRT-PCR.

<p>The transcript abundance from Transcriptome Profiling data is shown above each gene. Relative transcript levels are calculated by real-time PCR using alpha-tubulin as the standard. Three biological replicates were performed, and the data shown are typical results.</p

Histogram presentation of GO classification.

<p>A total of 35,268 (32.15%), 43,713 (39.84%) and 21,334 (19.45%) unigenes of <i>S. europaea</i> were classified into 44 terms from three ontologies involved in biological processes, cellular components and molecular function, respectively. The <i>right y axis</i> indicates the number of genes in a category. The <i>left y axis</i> indicates the percentage of a specific category of genes in the main category.</p

Se200S-vs-SeCKS differentially expressed genes.

<p>DEGs were filtered using FDR ≤0.001 and Log2Ratio ≤1 as a threshold. Red spots represent upregulated genes, and green spots indicate downregulated genes. Blue represents those genes that did not show obvious changes between the Se200S and SeCKS samples.</p

ChIP-seq and RNA-seq identify CdpR regulons in <i>P</i>. <i>aeruginosa</i> genome.

<p><b>(A)</b> The positions of the CdpR-binding peaks are presented in a pie chart. <b>(B)</b> CdpR binds to the inside sequence of PA0440. <b>(C)</b> Pie chart presents the percentages of CdpR targets with functional categories defined in the <i>Pseudomonas</i> database (<a href="http://pseudomonas.com" target="_blank">http://pseudomonas.com</a>). <b>(D)</b> The most highly significant motif identified by ChIP-seq using the MEME tool is shown. The height of each letter represents the relative frequency of each base at different position in the consensus sequence.</p